The present invention provides Methyl- (or Mutant-) Differential Display (MDD) methods and nucleic acid probes for detecting mutations and/or the methylation patterns of nucleic acids. Genes are frequently not methylated in the cells where they are expressed but are methylated in cell types where they are not expressed. Moreover, tumor cell DNA is frequently methylated to a different extent and in different regions than is the DNA of normal cells. The present invention is used for identifying which regions of the genome are methylated or mutated in different cell types, including cancerous cell types. The present invention also is used for identifying whether a tissue sample has a methylation or mutation pattern which is normal or like that of known cancer cells. The present invention can be used to identify aberrant expression of known or unknown genes. Such aberrant expression may occur at an incorrect location or an incorrect time, i.e. tissue or developmental specificity is lost. Genes and genomic regions discovered according to the present invention are useful for identifying products, e.g. mRNA, protein or fragments thereof, which are aberrantly expressed.
DNA is often methylated in normal mammalian cells. For example, DNA is methylated to determine whether a given gene will be expressed and whether the maternal or the paternal allele of that gene will be expressed. (Little and Wainwright, 1995). While methylation is known to occur at CpG sequences, only recent studies indicate that CpNpG sequences may be methylated (Clark et al., 1995). Methylation at CpG sites has been much more widely studied and is better understood.
Methylation occurs by enzymatic recognition of CpG and CpNpG sequences followed by placement of a methyl (CH3) group on the fifth carbon atom of a cytosine base. The enzyme that mediates methylation of CpG dinucleotides, 5-cytosine methyltransferase, is essential for embryonic developmentxe2x80x94without it embryos die soon after gastrulation. It is not yet clear whether this enzyme methylates CpNpG sites (Laird and Jaenisch, 1994).
When a gene has many methylated cytosines it is less likely to be expressed (Willson, 1991). Hence, if a maternally-inherited gene is more highly methylated than the paternally-inherited gene, the paternally-inherited gene will generally give rise to more gene product. Similarly, when a gene is expressed in a tissue-specific manner, that gene will often be unmethylated in the tissues where it is active, but will be highly methylated in the tissues where it is inactive. Incorrect methylation is thought to be the cause of some diseases including Beckwith-Wiedemann syndrome and Prader-Willi syndrome (Henry et al., 1991; Nicholls et al., 1989).
The methylation patterns of DNA from tumor cells are generally different than those of normal cells (Laird, 1994). Tumor cell DNA is generally undermethylated relative to normal cell DNA, but selected regions of the tumor cell genome may be more highly methylated than the same regions of a normal cell""s genome. Hence, detection of altered methylation patterns in the DNA of a tissue sample is an indication that the tissue is cancerous. For example, the gene for Insulin-Like Growth Factor 2 (IGF2) is hypomethylated in a number of cancerous tissues, such as Wilm""s Tumors, rhabdomyosarcoma, lung cancer and hepatoblastomas (Rainier et al., 1993; Ogawa et al., 1993; Zhan et al., 1994; Pedone et al., 1994; Suzuki et al., 1994; Rainier et al., 1995).
The present invention is directed to a method of detecting differential methylation at CpNpG sequences by cutting test and control DNAs with a restriction enzyme that will not cut methylated DNA, and then detecting the difference in size of the resulting restriction fragments.
While methylation-sensitive restriction enzymes have been used for observing differential methylation in various cells, no commercial assays exist for use on human samples because differentially methylated sequences represent such a minute proportion of the human genome that they are not readily detected. The human genome is both highly complex, in that it contains a great diversity of DNA sequences, and highly repetitive, in that it contains a lot of DNA with very similar or identical sequences. The high complexity and repetitiveness of human DNA confounds efforts at detecting and isolating the minute amount of differentially methylated DNA which may be present in a test sample. The present invention remedies this detection problem by providing new procedures for screening a selected subset of the mammalian genome which is most likely to contain genetic functions.
The present invention provides techniques for detecting and isolating differentially methylated or mutated segments of DNA which may be present in a tissue sample in only minute amounts by using one or more rounds of DNA amplification coupled with subtractive hybridization to identify such segments of DNA. DNA amplification has been coupled with subtractive hybridization in the Representational Difference Analysis (RDA) procedures disclosed in U.S. Pat. No. 5,436,142 to Wigler et al. and Lisitsyn et al. (1993). However, for the subtractive hybridization step of such RDA procedures to proceed in a reasonable time and with reasonable efficiency, only a subset of the genome can be examined. To accomplish this necessary reduction in the complexity of the sample DNA, Wigler et al. and Lisitsyn et al. disclose cutting DNA samples with restriction enzymes that cut infrequently and randomly. However, selection of enzymes which randomly cut the genome means that the portion of the genome which is examined is not enriched for any particular population of DNA fragments. Thus, when RDA is used, only a random subset of the human genome, which includes repetitive elements, noncoding regions and other sequences which are generally not of interest, can be tested in a single experiment.
In contrast, the present invention is directed to methods which use enzymes that cut frequently and that specifically cut CG-rich regions of the genome. These enzymes are chosen because CG-rich regions of the genome are not evenly distributed in the genomexe2x80x94instead, CG-rich regions are frequently found near genes, and particularly near the promoter regions of genes. This means that the proportion of the genome that is examined by the present methods will be enriched for genetically-encoded sequences as well as for regulatory sequences. Moreover, unlike the RDA method, the present methods selectively identify regions of the genome which are hypomethylated or hypermethylated by using enzymes which specifically cut non-methylated CG-rich sequences. The present invention therefore represents an improvement over RDA methods because of its ability to select DNA fragments which are likely to be near or to encode genetic functions.
The present invention provides probes and methods of detecting whether a CNG triplet is hypomethylated or hypermethylated in a genomic DNA present in a test sample of cell.
The present invention provides a method of detecting whether a CNG triplet is hypomethylated or hypermethylated in a genomic DNA present in a test sample of cells which includes:
a) isolating genomic DNA from a control sample of cells and a test sample of cells to generate a control-cell DNA and a test-cell DNA;
b) cleaving the control-cell DNA and the test-cell DNA with a master restriction enzyme to generate cleaved control-cell DNA and cleaved test-cell DNA;
c) preparing a probe from a DNA isolated by the present methods, for example, a DNA selected from the group consisting of SEQ ID NO:7-10;
d) hybridizing the probe to the cleaved control-cell DNA and the cleaved test-cell DNA to form a control-hybridization complex and a test-hybridization complex; and
e) observing whether the size of the control-hybridization complex is the same as the size of the test-hybridization complex;
wherein the master restriction enzyme cleaves a nonmethylated CNG DNA sequence but does not cleave a methylated CNG DNA sequence.
Similarly, the present invention provides a method of detecting whether a DNA site is mutated in a genomic DNA present in a test sample of cells which includes steps a) through e) above but where a detector restriction enzyme is used instead of the master restriction enzyme, wherein the detector restriction enzyme does not cleave a mutated DNA site but does cleave a corresponding nonmutated DNA site.
The present invention also provides methods of isolating probes to detect hypomethylation or hypermethylation in a CNG triplet of DNA. In particular, the present invention provides a method of isolating a probe to detect hypomethylation in a CNG triplet of DNA which includes:
a) cleaving a tester sample of genomic DNA with both a master restriction enzyme and a partner restriction enzyme to generate a cleaved tester sample;
b) ligating a first set of adaptors onto master enzyme cut DNA ends of the cleaved tester sample to generate a first-tester amplification template;
c) amplifying the first-tester amplification template to generate a first-tester amplicon by in vitro DNA amplification using primers that hybridize to the first set of adaptors;
d) cleaving off the first adaptors from the first-tester amplicon and ligating a second set of adaptors onto DNA ends of the first-tester amplicon to generate a second-adaptor-tester which has second adaptor ends;
e) melting and hybridizing the second-adaptor-tester with about a 10-fold to about a 10,000-fold molar excess of a driver DNA to generate a mixture of tester-tester product and tester-driver product;
f) adding nucleotides onto DNA ends present in the mixture to make a blunt-ended tester-tester product and a blunt-ended tester-driver product;
g) amplifying the blunt-ended tester-tester product and the blunt-ended tester-driver product by in vitro DNA amplification using primers that hybridize to second adaptor ends to generate a second-tester amplicon;
f) isolating a discrete DNA fragment from the second tester amplicon as a probe to detect the hypomethylation or the hypermethylation in a CNG triplet of DNA;
wherein the master restriction enzyme cleaves a nonmethylated CNG DNA sequence but does not cleave a methylated CNG DNA sequence;
wherein the partner restriction enzyme cleaves DNA to produce DNA fragments with a complexity of about 5% to about 25% of the genomic DNA in a size range which can be amplified by a DNA amplification enzyme; and
wherein the driver DNA is cut with both the master restriction enzyme and the partner restriction enzyme and amplified using primers that recognize DNA ends cut by the master restriction enzyme.
Similarly, when the present invention provides a method of isolating a probe to detect hypermethylation in a CNG triplet of DNA, normal DNA is used instead of the tester DNA and the normal amplicon is annealed and hybridized with a molar excess of tester driver DNA.
Moreover the present methods are used to isolate probes to detect a mutation in a test sample of genomic DNA by using a detector enzyme instead of a partner restriction enzyme, wherein the detector restriction enzyme cleaves a normal DNA site but not a mutant DNA site.
The present invention further provides a kit for detecting hypomethylation in a CNG triplet of DNA which is present in a test tissue sample which includes a DNA or a probe isolated by the methods of the present invention, for example, a DNA having SEQ ID NO:7-10.
The present invention provides nucleic acid sequences which are differentially methylated in human breast and ovarian cancer tissues, as compared to normal tissue.
The present invention provides nucleic acid sequences which are useful as probes for determining the methylation state of genomic DNA sequences which are differentially methylated in tumor tissues.
Similarly, the present invention provides nucleic acid sequences which are useful as probes for the detection of mRNA expression, as well as oligonucleotide sequences which can be used to amplify and thus detect normal and aberrant expression of particular mRNA species.
The DNA sequences provided herein may also be expressed in host cells for the production of the encoded protein or protein fragments.
The proteins and protein fragments expressed from the nucleotide sequences disclosed herein are useful for the production of monoclonal antibodies (Mabs). Such Mabs have a variety of uses including, but not limited to, detection of normal or aberrant protein expression in human tissues and fluids.
Antibodies of the invention bind selectively to tsp50 protein wherein tsp50 protein comprises SEQ ID NO:16 from about amino acid residue number 1 to about amino acid residue number 385. The term xe2x80x9cantibodyxe2x80x9d is used herein to include complete antibodies (e.g., bivalent IgG, pentavalent IgM) as well as fragments of antibodies which contain an antigen binding site. Such fragment include, but are not limited to Fab, F(abxe2x80x2)2, Fv and single chain Fv (scFv) fragments. Such fragments may or may not include antibody constant domains. For example, Fab""s lack constant domains which are required for complement fixation. scFvs are composed of an antibody variable light chain (VL) linked to a variable heavy chain (VH) by a flexible linker. scFvs are able to bind antigen and can be rapidly produced in bacteria. The invention includes antibodies and antibody fragments which are produced in bacteria and in mammalian cell culture. An antibody obtained from a bacteriophage library can be a complete antibody or an antibody fragment. Generally, the domains actually present in such a library are heavy chain variable domains (VH) and light chain variable domains (VL) which together comprise Fv or scFv, with the addition, in some cases, of a heavy chain constant domain (CH1) and a light chain constant domain (CL). The four domains (i.e., VH-CH1 and VL-CL) comprise an Fab. Complete antibodies are obtained from such a library by replacing missing constant domains once a desired VH-VL combination has been identified.
Antibodies of the invention can be monoclonal antibodies (Mab) or polyclonal antibodies. Antibodies of the invention which are useful for in vitro detection of tsp50 protein can be from any source, and in addition may be chimeric antibodies which comprise sequences of amino acids from two or more sources. Sources include but are not limited to a mouse or a rat or a human. Preferred antibodies for use in vivo (i.e., detection or treatment of cancer) have reduced antigenicity in humans, and more preferably are not antigenic in humans. Chimeric antibodies for use in vivo contain human amino acid sequences and include humanized antibodies which are non-human antibodies substituted with sequences of human origin to reduce or eliminate immunogenicity, but which retain the binding characteristics of the non-human antibody. Particularly preferred antibodies are human antibodies.
Polyclonal antibodies can be obtained directly from the serum of animals immunized with tsp50. Human polyclonal antibodies can be obtained from transgenic animals which comprise human Ig genes. Human antibodies can also be obtained from combinatorial libraries comprising cells expressing combinations of human heavy and light chains. Human antibodies have human heavy chains and human light chains. Human antibodies also include variants which have been created to, for example, improve antigen binding characteristics. In a normal immune response, this occurs through somatic mutation and selection. In vitro, this is accomplished by mutation of one or more residues, followed by screening for antibodies having preferred binding characteristics. Preferred binding characteristics are improved affinity and/or improved specificity.
Bacteriophage libraries used to obtain antibodies of the invention comprise combinations of rearranged Ig genes from mature mammalian B cells. Animals, including animals comprising human Ig genes, from which these libraries are developed may be first immunized with tsp50 to increase the proportion of the library which is reactive with tsp50. Human antibodies of the invention can therefore be obtained from bacteriophage libraries made from immunized transgenic animals comprising human Ig genes, which animals may first have been immunized with tsp50. Alternatively, human antibodies can be obtained from bacteriophage libraries developed directly from human immune system cells. Human antibodies further comprise those which have been obtained by these or similar methods which are then modified to improve their binding characteristics.
The invention also provides chimeric anti-tsp50 antibodies which have binding sites derived from non-human anti-tsp50 antibodies. To minimize immunogenicity in humans, chimeric anti-tsp50 antibodies of the invention comprise non-binding site amino acids which have been substituted with human sequences. The humanized regions of chimeric anti-tsp50 antibodies can be heavy and light chain constant domains. The humanized regions can also be framework regions of variable domains.
The humanized regions can also be constant region and variable region amino acids which are exposed on the surface of the antibody molecule. Chimeric and humanized antibodies of the invention further comprise chimeric antibodies and humanized antibodies which have been modified to improve their binding characteristics. Improved binding can mean greater affinity and/or greater specificity. Such modification can be the result of, for example, in vitro mutation and selection.
Antibodies of the invention may be linked to an anti-tumor agent or a detectable label. This allows the antibody to target the anti-tumor agent or detectable label to the tumor. Thus, antibodies of the invention can be suitable for use in a method of treatment of the human body or in a method of diagnosis applied directly to the human body, or in a method of diagnosis applied to samples of human tissues or body fluids obtained from the human body. A review of the use of antibodies in diagnosis and therapy is provided by Waldmann (1991).
The invention includes pharmaceutical compositions which comprise the aforementioned antibodies and antibody conjugates.